Proteases are commonly added to animal feed in order to increase the protein digestibility of the feed. In some cases, the protease is added to the feed prior to the pelleting process, which involves heating the feed mixture to high temperatures (70° C.-95°+C). In other instances, the protease enzyme is sprayed onto and/or mixed into the feed. Either way, it is often desirable to measure the amount of protease activity in the feed product to make sure that the protease was in fact added, that the protease was added in the correct quantity, and that the protease survived the pelleting and/or mixing process.
Numerous methods are known for the detection of protease activity in samples and the methods are often based on the use of natural protein substrates, synthetic peptide substrates, or peptide analog substrates labeled with a chromophore, a flurophore, or a radioisotope to detect the protease activity. However, the problem in measuring protease activity that has been added to feed products is that naturally occurring components of feed, including endogenous protein substrates in the feed, can compete with the labeled substrate being used to detect and measure the protease activity. This issue creates a significant challenge to testing protease activity in feed.
For example, PROTAZYME AK tablets from MEGAZYME, INC. are useful for quantifying the activity of pure proteases such as VERSAZYME (BIORESOURCE INTERNATIONAL, INC.), RONOZYME PROACT (RONOZYME, INC.), and AXTRA 102 TPT XAP (DANISCO, INC.) prior to addition as feed additives. However, the manufacture acknowledges the limitations of using the PROTAZYME AK substrate to measure protease activity once it has been added to feed (see http://secure.megazyme.com/files/FAQ%27s/T-PRAK1000—1204_FAQ.pdf). In an attempt to get around the issue of the feed inhibiting the protease activity measurement, existing methods require steps to first extract the added protease from the feed and to then separate the insoluble feed from the protease-containing soluble extract. The extraction and separation steps add a level of complexity to the in-feed assay that results in the need for sophisticated equipment such as for example a grinder, a micropipettor, a spectrophotometer, and a centrifuge. The use of such equipment increases the cost of the assay and also usually requires the sample to be sent out from the feed-processing facility to a laboratory that is set up to perform the assay. This then dramatically increases the time needed to obtain the test results.
Accordingly, there remains an unmet need for an in-feed/in-food protease assay that can be performed on-site and without the need for sophisticated equipment. The presently disclosed subject matter provides such an assay.